ABCG1 Ameliorates Diabetic Vascular Disease Through Regulating The Function Of Endothelial Progenitor Cell

Objectives:Diabetes mellitus（DM）is a complex metabolic disease and accelerated vascular disease is the principal cause of death and disability in patients with diabetes mellitus.Previous studies have revealed that endothelial progenitor cells（EPCs）play an important role in maintaining endothelial integrity.However,reduction of circulating EPCs levels and impaired EPCs function have been reported in diabetic patients and animals.Thus,interventions that enhance EPC number and function in diabetes may prove to be particularly benefit for clinical thereby.Studies shows that genes that regulate cellular cholesterol hemostasis are associated with EPCs biological function.Although ABCG1 has a critical role in maintain cholesterol hemostasis,little was known about its role in EPCs.Methods:Firstly,we induced I type diabetes model by STZ and performed wire injuryin the carotid artery of wild type（WT）C57/BL6 mice.To explore the role of endothelial progenitor cells in re-endothelialization in diabetes,we injected Dil-ac-LDL labeled BM-EPCs from Tg mice and littermate WT mice through tail vein three hours after carotid artery injury.Re-endothelialization was quantified by enface Evans blue staining at 3days after injury.Next,we assessed the effect of ABCG1 on EPC number and function in diabetic mice.The levels of circulating EPCs were detected via flow cytometry.Furthermore,bone marrow derived EPCs were used for migration and tube formation assay.The phospholation level of eNOS and Lyn kinase was detected by western blot.Results:It demonstrated EPC transplantation restore injuried arteries and there were more labeled Tg EPCs recruited in the injuryed artery.We found that the number of CD34⁺/VEGFR2⁺positive cells was significantly lower in diabetic mice than WT control mice.Overexpression of ABCG1 can increase circulating EPCs level and reverse the reduction effect induced by diabetes in Tg mice.A dramatic increase of migration was observed in transgenic EPCs and abolished the antimigratory effect of diabetes.We measured the effects of ABCG1 on EPCs migration and tube formation capacity in high glucose conditions.The results indicated that the migratory and tube formation capacity was significantly reduced after treatment with 25 mM D-glucose for 48 hour.However,transgenic EPCs restore the inhibitory effect induced by high glucose.Ectopic expression of ABCG1 by adenovirus increased the expression levels of CD31 and KDR,whereas the expression levels of CD34and c-kit were reduced;Thus,overexpression ABCG1 promoted EPCs differentiation.Meanwhile,ABCG1-overexpressing EPCs showed an increased migratory capacity and tubule network formation to a level that similar with VEGF stimulation.However,ABCG1 knock-down inhibited EPCs differentiation.Furthermore,migration activity was significantly reduced in ABCG1-silenced EPCs compared with control EPCs.Moreover,silencing of ABCG1 led to significantly deterioration of the capillary-like tube formation of EPCs.In vivo angiogenesis assay,matrigel containing EPCs from WT and transgenic mice was injected subcutaneously into both WT mice and transgenic mice.Tg-EPCs promoted tubule network formation in WT and transgenic mice.In addition,the ABCG1-tg mice displayed more capillary-like structure than WT mice.Moreover,EPCs exhibited a poor tube formation capacity after knocking down ABCG1expression.We also found the phospholation level of eNOS protein increased after ad-ABCG1 treatment in EPCs,but reduced by ABCG1 siRNA.To indentify wether ABCG1 affect EPC function trough regulating eNOS activity.We isolated BM-EPCs from eNOS-/-knock out mice and treated with ad-ABCG1 adenovirus.The migratory capacity and tubule network formation was significantly reduced in eNOS^-/-EPCs,however,ABCG1 overexpression can not reverse the inhibitory effects by eNOS deficiency.Next,we found a significantly increase in Lyn kinase phosphorylation in response to high expression of ABCG1.After treatment with cyclodextrin to remove cellular cholesterol,phosphorylation of Lyn kinase was significantly increased,but cholesterol-cyclodextrin complexes abolished the phosphorylation.Also,an inhibitor of Lyn kinase--SU6656,significantly reduced eNOS phosphorylation.Treatment of EPCs from wild-type or transgenic mice with Lyn siRNA reduced eNOS activity through akt signal pathways.These findings indicated that ABCG1 regulated EPC function by activating eNOS through Lyn/Akt pathway.Conclusions:ABCG1 improves EPC migration and angiogenesis and promotes re-endothelialization of injured ateries in diabetic mice.In addition,ABCG1 can enhance the number and function of EPCs to repair the vascular injury induced by diabetes.Our findings indicate that ABCG1 might be a new therapeutic targets of diabetes associated vascular disease.